Indole-containing and combretastatin-related anti-mitotic and anti-tubulin polymerization agents

ABSTRACT

Trimethoxyphenyl substituted indole ligands have been discovered which demonstrate impressive cytotoxicity as well as a remarkable ability to inhibit tubulin polymerization. Such compounds as well as related derivatives are excellent clinical candidates for the treatment of cancer in humans. In addition, certain of these ligands, as pro-drugs, may well prove to be tumor selective vascular targeting and destruction chemotherapeutic agents or to have anti-angiogenesis activity resulting in the selective prevention and/or destruction of tumor cell vasculature.

BACKGROUND OF THE INVENTION

Tubulin is currently among the most attractive therapeutic targets innew drug design for the treatment of solid tumors.^(1c) The heraldedsuccess of vincristine and taxol along with the promise ofcombretastatin A-4 (CA-4) prodrug and dolastatin 10, to name just a few,have firmly established the clinical efficacy of these antimitoticagents for cancer treatment.

An aggressive chemotherapeutic strategy toward the treatment ofsolid-tumor cancers continues to rely on the development ofarchitecturally new and biologically more potent anti-tumor,anti-mitotic agents which mediate their effect through a direct bindinginteraction with tubulin. A variety of clinically-promising compoundswhich demonstrate potent cytotoxicity and antitumor activity are knownto effect their primary mode of action through an efficient inhibitionof tubulin polymerization.¹ This class of compounds undergoes an initialinteraction (binding) to the ubiquitous protein tubulin which in turnarrests the ability of tubulin to polymerize into microtubules which areessential components for cell maintenance and division.² Duringmetaphase of the cell cycle, the nuclear membrane is broken down and thecytoskeletal protein tubulin is able to form centrosomes (also calledmicrotubule organizing centers) and through polymerization anddepolymerization of tubulin the dividing chromosomes are separated.Currently, the most recognized and clinically useful members of thisclass of antimitotic, antitumor agents are vinblastine and vincristine³along with taxol.⁴ Additionally, the natural products rhizoxin,⁵combretastatin A-4 and A-2,⁶ curacin A,¹ podophyllotoxin,⁷ epothilones Aand B,⁸ dolastatin 10⁹ and welwistatin¹⁰ (to name just a few) as well ascertain synthetic analogues including phenstatin,¹¹ the2-styrylquinazolin-4(3H)-ones (SQO),¹² and highly oxygenated derivativesof cis- and trans-stilbene¹³ and dihydrostilbene are all known tomediate their cytotoxic activity through a binding interaction withtubulin. The exact nature of this binding site interaction remainslargely unknown , and definitely varies between the series of compounds.Photoaffinity labeling and other binding site elucidation techniqueshave identified several key binding sites on tubulin: colchicine site,vinca alkaloid site, and a site on the polymerized microtubule to whichtaxol binds.^(la.14)

SUMMARY OF THE INVENTION

An important basic and essential aspect of this work requires a detailedunderstanding, on the molecular level, of the “small molecule” bindingdomain of both the α and β subunits of tubulin. The tertiary structureof the α, β tubulin heterodimer was reported earlier this year byDowning and co-workers at a resolution of 3.7 Å using a technique knownas electron crystallography.¹⁵ This brilliant accomplishment culminatesdecades of work directed toward the elucidation of this structure andshould facilitate the identification of small molecule binding sites,such as the colchicine site, through techniques such as photoaffinityand chemical affinity labeling.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates 3-(3′, 4′,5′-trimethoxybenzoyl)-2-(4′-methoxyphenyl)-6-methoxybenzo[b]thiophene.

FIG. 2 illustrates 2-(3′, 4′,5′-trimethoxybenzoyl)-3-(4′-methoxyphenyl)-6-methoxybenzo[b]furan.

FIG. 3 illustrates benzo[b]thiophene Phenol (BBT-OH).

FIG. 4 illustrates benzo[b]thiophene prodrug (BBT-P).

FIG. 5 illustrates in vivo biological data for benzo[b]thiophene prodrug(BBT-P).

FIG. 6 illustrates a synthetic route for preparation of phenylindolederivatives.

FIG. 7 illustrates a COSY NMR for 2-phenyl indole (aromatic region) acompound 31.

FIG. 8 illustrates a cyclized isomer without aryl migration (no evidencefor its formation).

FIG. 9 illustrates a preparation of 2-phenylindole 31 in a one-potreaction.

FIG. 10 illustrates a designed synthetic route for preparation ofindole-based analog.

FIG. 11 illustrates a preparation of indole-based analog.

FIG. 12 illustrates a synthesis of indole-based disodium prodrug salt.

FIG. 13 illustrates another synthesis of indole-based disodium prodrug.

FIG. 14 illustrates another synthesis of indole-based disodium prodrug.

FIG. 15 illustrates a synthesis of indole based phosphoramidate prodrug.

FIG. 16 illustrates another synthesis of indole-based disodium prodrugsalt.

FIG. 17A illustrates a combretastatin A-4 pro-drug.

FIG. 17B illustrates a phosphoramidate analog 10.

FIG. 18 illustrates a synthesis of phosphoramidate 10.

FIG. 19 illustrates a model system used for phosphoramidate synthesis.

FIG. 20 illustrates a synthesis of phosphoramidate 10 from from(Z)-3′-nitro combrestastatin analog 7B.

FIG. 21 illustrates substituted 4-methoxyindole amines and/or phenols.

FIG. 22 illustrates substituted 4-methoxyindole phosphate ester moietiesand phosphoramidates.

FIG. 23 illustrates further substituted 4-methoxyindole phosphate estermoieties and phosphoramidates.

FIG. 24 illustrates substituted 6-methoxyindole amines and/or phenols.

FIG. 25 illustrates substituted 6-methoxyindole phosphate ester moietiesand phosphoramidates.

FIG. 26 illustrates substituted 6-methoxyindole phosphate ester moietiesand phosphoramidates.

FIG. 27 illustrates substituted 4-methoxy-3-arylindole amines and/orphenols.

FIG. 28 illustrates substituted 4-methoxy-3-arylindole phospate-moieties and phosphoramidates.

FIG. 29 illustrates further substituted 4-methoxy-3-arylindole phospatemoieties and phosphoramidates.

FIG. 30 illustrates 2-(4′-Methoxyphenyl)-3-(3″, 4″,5″-trimethoxybenzoyl)-4- methoxyindole.

FIG. 31 illustrates 2-(3′, 4′,5′-Trimethoxybenzoyl)-3-(4″-methoxyphenyl)-6- methoxyindole.

FIG. 32 illustrates 2-(3′, 4′,5′-Trimethoxybenzoyl)-3-(4″-methoxyphenyl)-4- methoxyindole.

FIG. 33 illustrates Disodium2-(3′-phosphoramidate4′-methoxyphenyl)-3-(3″, 4″, 5″-trimethoxybenzoyl)-6-methoxyindole.

FIG. 34 illustrates 2-(3′-Hydroxy-4′-methoxyphenyl)-3-(3″, 4″,5″-trimethoxybenzoyl)- 4-methoxyindole.

FIG. 35 illustrates 2-(3′-Amino-4′-methoxyphenyl)-3-(3″, 4″,5″-trimethoxybenzoyl)- 4-methoxyindole.

FIG. 36 illustrates Disodium2-[(4′-methoxyphenyl)-3′-O-phosphate]-3-(3″, 4″,5″trimethoxybenzoyl)-4-methoxyindole.

FIG. 37 illustrates2-(3′-Diethylphosphoramidate-4′-methoxyphenyl)-3-(3″, 4″, 5″-trimethoxybenzoyl)-4-methoxyindole.

FIG. 38 illustrates Disodium2-(3′-phosphoramidate-4′-methoxyphenyl)-3-(3″, 4″, 5″-trimethoxybenzoyl)-4-methoxyindole.

FIG. 39 illustrates2-(3′,4′,5′-trimethoxybenzoyl)-3-(3″-hydroxy-4″-methoxyphenyl)-6-methoxyindole.

FIG. 40 illustrates2-(3′,4′,5′-trimethoxybenzoyl)-3-(3″-amino-4″-methoxyphenyl)-6- 10methoxyindole.

FIG. 41 illustrates Disodium2-(3′,4′,5′-trimethoxybenzoyl)-3-[(4″-methoxyphenyl-3 ″-O-phosphate)]-6-methoxyindole.

FIG. 42 illustrates2-(3′,4′,5′-trimethoxybenzoyl)-3-[(4″-methoxyphenyl-3″-diethylphosphoramidate)]-6-methoxyindole.

FIG. 43 illustrates Disodium2-(3′,4′,5′-trimethoxybenzoyl)-3-[(4″-methoxyphenyl-3″-phosphoramidate)]-6-methoxyindole.

FIG. 44 illustrates2-(3′,4′,5′-trimethoxybenzoyl)-3-(3″-hydroxy-4″-methoxyphenyl)-4-methoxyindole.

FIG. 45 illustrates2-(3′,4′,5′-trimethoxybenzoyl)-3-(3″-amino4″-methoxyphenyl)-4- 20methoxyindole.

FIG. 46 illustrates Disodium2-(3′,4′,5′-trimethoxybenzoyl)-3-[(4″-methoxyphenyl-3″- O-phosphate)]-4-methoxyindole.

FIG. 47 illustrates2-(3′,4′,5′-trimethoxybenzoyl)-3-[(4″-methoxyphenyl-3″-diethylphosphoramidate)]-4-methoxyindole.

FIG. 48 illustrates Disodium2-(3′,4′,5′-trimethoxybenzoyl)-3-[(4″-methoxyphenyl-3 ″-phosphoramidate)]-4-methoxyindole.

FIG. 49 illustrates substituted 3-phosphoramidate derivatives ofcombretastatin A-4.

FIG. 50 illustrates Disodium (Z)- I-[(4′-methoxyphenyl)-3′-phosphoramidate]-2-(3″,4″,5″-trimethoxyphenyl)ethene

FIG. 51 illustrates substituted 3-phosphoramidate salts ofcombretastatin A-4.

DETAILED DESCRIPTION OF THE INVENTION

We have developed a working hypothesis suggesting that the discovery ofnew antimitotic agents may result from the judicious combination of amolecular template (scaffold) which in appropriately substituted form(ie. phenolic moieties, etc.) interacts with estrogen receptor (ER),suitably modified with structural features deemed imperative for tubulinbinding (arylalkoxy groups, certain halogen substitutions, etc.). Themethoxy aryl functionality seems especially important for increasedinteraction at the colchicine binding site in certain analogs.¹⁶ Uponformulation of this hypothesis concerning ER molecular templates, ourinitial design and synthesis efforts centered on benzo [b]thiopheneligands modeled after raloxifene, the selective estrogen receptormodulator (SERM) developed by Eli Lilly and Co.¹⁷ Our initial studiesresulted in the preparation of a very active benzo[b]thiophene-basedantitubulin agent. ¹⁸⁻²¹ In further support of our hypothesis, recentstudies have shown that certain estrogen receptor (ER) binding compoundsas structurally modified estradiol congeners (2-methoxyestradiol, forexample) interact with tubulin and inhibit tubulin polymerization.²²Estradiol is, of course, perhaps the most important estrogen in humans,and it is intriguing and instructive that the addition of the methoxyaryl motif to this compound makes it interactive with tubulin. It isalso noteworthy that 2-methoxyestradiol is a natural mammalianmetabolite of estradiol and may play a cell growth regulatory roleespecially prominent during pregnancy.

The design premise that molecular skeletons of traditional estrogenreceptor (ER) binding compounds can be modified with structural motifsreminiscent of colchicine and combretastatin A-4 to produce inhibitorsof tubulin polymerization has been validated by the benzo[b]thiopheneand benzol[b]furan classes of new antimitotic agents. ¹⁸⁻²¹ The leadcompounds in each series (FIGS. 1 and 2), demonstrate remarkablebiological activity against a variety of human cancer cell lines. Forexample, the 3,4,5-trimethoxybenzo[b]thiophene (FIG. 1) demonstratespotent cytotoxicity and inhibition of tubulin polymerization. In the NCI60 cell line panel,²³ this compound produces a mean panel G1₅₀=2.63×10⁻⁷M (see Table I).

Inhibition of tubulin polymerization by 3-(3′, 4′,5′-trimethoxybenzoyl)-2-(4′- methoxyphenyl)-6-methoxybenzo[b]thiophene.50% inhibition of the maximum tubulin assembly rate with 1.1 μM drugsame assay with conbretastatin A4 gives a value of 0.73 μM.

Human cancer cell line studies (in vitro) by 3-(3′, 4′,5′-trimethoxybenzoyl)-2-(4′- methoxyphenyl)-6-methoxybenzo[b]thiophene.TABLE I Inhibition of tubulin polymerization by 2-(3′, 4′, 5′-trimethoxybenzoyl)-3-(4′-methoxyphenyl)-6-methoxy- benzo[b]furan. IC50 =2.1 pM (totally flat at 4 pM). Human cancer cell line studies (in vitro)by 2-(3′, 4′, 5′- trimethoxybenzoyl)-3-(4′-methoxyphenyl)-6-methoxybenzo[b]furan. Type of Cancer Cell Line Cancer Cell Line GI₅₀(uglmL) Pancreas - adn BXPC-3 0.038 Neuroblast SK-N-SH 0.025 Thyroid caSW1736 0.047 Lung-NSC NCI-H460 0.041 Pharynx-sqam FADU 0.035 ProstateDU-145 0.062

In addition, the phenolic derivative of the3,4-5-trimethoxybenzo[b]thiophene compound (FIG. 3) has pronouncedcytotoxicity and demonstrates outstanding inhibition of tubulinpolymerization³⁶ and the pro-drug disodium phosphate salt form of thiscompound (FIG. 4) demonstrates in vitro and in vivo cytotoxicity as avascular targeting and destruction agent (which includes a component oftubulin binding (phenolic form of drug) ^(36,37) and subsequentinhibition of tubulin polymerization).

Initial in vivo studies are very encouraging (see FIG. 5). Female scidmice were single dose ip administered with CA-4P, and benzo[b]thiophenephosphate prodrug at 400 mg/kg (i.e. MDT of CA-4P) after one week ofMHEC inoculation (1×10⁻⁶/mouse). Studies were carried out through acollaboration with Professors Ronald W. Pero and Klaus Edvardsen,University of Lund, Sweden (Note: PbT Prodrug 20 is the same compoundthat is referred to as BBT-P).

Based on these promising research results, our interest in designing anindole based antimitotic agent was initiated, and a synthetic route(Schemes 1-4, see FIGS. 3 A-D) was designed according to the synthesisof the benzo[b]thiophene derivatives.

The possibility clearly exists that some of the new indole-based ligandsdescribed herein, which are structurally related to combretastatin A-4,may also function through additional biological mechanisms involvinganti-angiogenic activity. Clearly the ability to selectively disrupt theblood-flow to developing tumor cells is a potential breakthrough in theever up-hill battle against cancer. Certain phenylindoles have beennoted for inhibiting tubulin polymerization.²⁷

A typical synthesis of indole-based ligand 33 is shown in FIGS. 6, 9 and11. Secondary amine 30 was prepared by treatment of m-anisidine and2-bromo-4methoxyacetophenone under basic condition (ethanolic potassiumhydroxide) at 0° C. Treatment of amine 30 with PPA resulted in theformation of two regioisomers. These isomers have poor solubility inEtOAc, CH₂Cl₂ and EtOH. Indole 31 was purified (from indole 32) bytrituration in acetone. The structure of this isomer was confirmed byNMR analysis. COSY NMR was taken in order to study, in detail, thecoupling relationship between the protons. The enlarged COSY spectrumfor the aromatic region of ligand 31 is shown in FIG. 5. This COSY NMRspectrum, shows a strong coupling between H^(a) and H^(b) which eachappear as a doublet. H^(c) is coupled by the proton attached to thenitrogen into a small doublet. H^(d) is coupled only by H^(e) into acorresponding doublet, while H^(e) is coupled both by an ortho coupling(H^(d)) and by a meta coupling (H^(f)) into a doublet of doubletpattern. H^(f) is coupled by H^(e) into a doublet. Further evidence ofthe formation of 2-phenyl indole 31 is the chemical shift of the protonH^(c) on the ring which contains nitrogen. Though computer modeling(ChemDraw Ultra 4.5), the theoretical chemical shift value of 6.4 ppm ispredicted for proton H^(c) (at the 3 position), which matches the peakshown in the actual NMR spectrum at 6.6 ppm. For the case where theproton is at the 2 position (FIG. 8), the chemical shift is predicted tobe 7.03 ppm, which does not match any peak in the spectrum that wasobtained. Based collectively on these studies, the formation of isomer31 is confirmed, and the migration of the methoxyphenyl system isevidenced. The other isomer (indole 32) is soluble in acetone and ismuch more difficult to obtain in pure form (see FIG. 6).

Alternatively, another synthetic methodology can also be applied to thepreparation of the desired 2-phenylindole. In 1984, Angerer andco-workers reported the synthesis of 2-phenylindoles in a one-potreaction sequence (FIG. 9) as a route toward the development of newtherapeutic agents for the treatment of endocrine disorders.²⁵

Following this procedure (FIG. 9), two arylindole regioisomers wereobtained in good yield. Recrystallization in EtOH afforded the desiredisomer, 2-phenylindole 31, as a white crystalline material.

In order to synthesize the indole-based analog 33, Friedel-Craftsacylation was carried out by treating indole 31 with3,4,5-trimethoxybenzoyl chloride in the presence of the Lewis-Acid AlCl₃( FIG. 10). The reaction did not work under the regular conditions andonly starting material was obtained following work-up. Attempts tomodify the reaction conditions by increasing the reaction temperature orusing other Lewis Acids, such as TiCl₄, proved futile as well. Startingmaterial was recovered in all cases. One possible explanation for thisresult is the fact that the nitrogen atom (containing a lone pair ofelectrons and an acidic proton) may disrupt the acylation process.According to this analysis, a Grignard reagent (ethylmagnesium bromide)was used to protect this nitrogen prior to the Friedel-Crafis acylationstep. Still, only starting material was obtained following the reaction.Therefore, a new synthetic approach was brought into this study.

In 1977, Inion and co-workers reported the synthesis of a variety ofaminoalkoxy4-benzoyl-3-indoles.²⁶ The benzoate indole product wasprepared by treatment of indole with the appropriate benzoyl chloridewith heating (130-150° C.). HCl is generated under these conditions. Asimilar synthetic approach was used in the synthesis of the desiredtrimethoxybenzoate indole ligand 33 (FIG. 11).

The precursor, indole 31, was mixed with trimethoxybenzoyl chloride.Since both reagents are solid, a solvent with a high boiling point wasneeded. 1,2-dichlorobenzene was chosen in this case since it has aboiling point of 180° C. Under these condition, indole 33 was obtainedin moderate yield following purification by flash column chromatographyand recrystallization. NMR spectroscopy suggests that the structure ofindole 33 is that indicated in FIG. 11.

Based on promising results obtained with benzo[b]thiophene andbenzofuran analogs, the preparation of phosphate salts is detailed inFIGS. 12-14, the preparation of analogs is detailed in FIGS. 15-16 andthe preparation of similar indole-based phosphate prodrug salts andphosphoramidate derivatives is detailed in FIGS. 21-51.

In addition to the phosphate ester prodrugs that are described in thisapplication for indole-based anti-mitotic agents, we have alsodiscovered that phosphorous based prodrug derivatives of the nitrogenanalog of combretastatin A-4 (CA-4) may have therapeutic advantages asselective tumor vasculature destruction agents. These compounds areprimarily phosphoramidate derivatives and related phosphate dianionsthat are assembled on the 3

amino substituent of the nitrogen analog of CA-4. Although we describetwo specific compounds and several obvious analogs, it should beapparent to anyone skilled in the art, that there are numerous othernitrogen phosphorous bond designs that might be assembled from the3-amino-combretastatin A-4 structure and that would display similarfunctionality as prodrugs for the selective destruction of tumorvasculature.

Further significance is given to new drugs that bind to the colchicinesite since it has recently been shown that combretastatin CA-4 alsodemonstrates anti-angiogenesis activity.²⁴ An emerging area of cancerchemotherapy centers on the development of both anti-angiogenesis drugswhich disrupt the new microvessel formation of developing tumors andvascular targeting and destruction agents which selectively target thevasculature of tumor cells while leaving healthy cells intact.Combretastatin CA-4P prodrug (FIG. 17A) is one of the leading newcandidates from among a relatively small collection of known worldcompounds which display this vaxcular targeting. Discovered by ProfessorGeorge R. Pettit (Arizona State University) from a willow tree(combretum caffrum) in South Africa in the 1970s, this compound iscurrently undergoing phase I clinical evaluation sponsored and licensedby OXiGENE, Inc.

Combretastatin A-4 (CA-4) is a potent inhibitor of tubulinpolymerization which binds to the colchicine site on β-tubulin.Interestingly, CA-4 itself does not demonstrate destruction of tumorvasculature, while CA-4 prodrug is very active in terms of tumorvasculature destruction. It is very likely that the phosphate esterportion of the prodrug undergoes dephosphorylation (perhaps through theaction of endothelial alkaline phosphatases) selectively at sites ofenhanced vascularization to reveal the potent CA-4 itself which destroysthe tumor cell through an inhibition of tubulin polymerization. Thedephosphorylation event takes place selectively at tumor cells sincetumor cells represent sites of prolific vascularization and alkalinephosphatases appear to be present at elevated concentrations in theendothelial cells lining tumor vasculature. This need for enhancedvascularization is not necessary for healthy cells. Hence, thisdual-mode reactivity profile is clearly important in order to targettumor cells selectively over healthy cells. This is a proposal which hasbeen advanced by Professor Ronald Pero (OXiGENE, Inc., University ofLund) for which a variety of strong evidence has been obtained.

Based in part on the good and promising biological results obtained forthe 3′-nitrogen analogs of combretastatin A-4, a phosphoramidate analoghas been prepared as a new combretastatin A-4 nitrogen prodrug (FIG.17B).

Phosphoramidate 10 below was obtained following the procedure reportedby Taylor and coworkers for unrelated aryl amines.²⁸ Treatment ofarylamine 7B with diethylchiorophosphite in anhydrous ether followed byoxidation with m-CPBA produced the phosphoramidate 10 in moderate yield(FIG. 18).

A previous attempt in the synthesis of the phosphoramidate analog 10utilized the methodology reported by Bilha Fisher and Larisa Sheihet.²⁹This methodology presents a phosphoramidate intermediate, which can beisolated from the reduction of nitro aryl compounds to the correspondingaryl amines using diethylchlorophosphite as a biphilic reagent. The(Z)-nitro combretastatin analog 7B was considered a viable startingmaterial for the synthesis of the phosphoramidate prodrug 10. Thisreaction was also tried using(Z)-1-(3′,4′,5′-trimethoxyphenyl)-2-(4″-nitrophenyl)ethene (synthesizedin a similar manner as the other combretastatin containing analogsreported previously) as a model system (FIG. 19). In neither case wasthe phosphoramidate product observed. It is thought that the presence ofmethoxy groups as strong electron donating substituents on the stilbenesystem disfavors the reaction (FIG. 20).

It should be obvious to anyone skilled in the art of phosphate ofphosporamidate chemistry that there are numerous other synthetic methodswhich can be employed to prepare phosphoramidates (such as 10) and theirrelated salts (-NHPO₃ ⁻²2Na^(+).) TABLE II³⁰ In vitro Human Cancer CellLine Study of Phosphoramidate Analog 10. GI₅₀, TGI, and LC₅₀ arereported as concentrations in μg/mL ND = Not determined Cell Type CellLine GI₅₀ TGI LC₅₀ Pancreas-a BXPC-3 1.5 × 10⁻¹ 5.7 × 10⁻¹ >10 OvarianOVCAR-3 1.9 × 10⁻¹ 8.6 × 10⁻¹ >10 CNS SF-295 2.4 × 10⁻¹ >10 >10 Lung-NSCNCI-H460 3.5 × 10⁻¹ >10 >10 Colon KM20L2 2.8 × 10⁻¹ 6.1 × 10⁻¹ >10Prostate −DU-145 2.6 × 10⁻¹ 2.6 × 10⁻¹ >10 Leukemia P388 3.1 × 10⁻¹ NDND

Biological evaluation (in vitro) suggests that the phosphoramidateprodrug 10 is less effective than the corresponding amine 8 (Table II).Pettit and co-workers reported a similar loss in biological activity invitro for the phosphate prodrugs of combretastatin A-4 and phenstatincompared to the original compounds (Table III).³¹ These results might beexplained by the bulkiness of the phosphorous group and its sterichindrance toward binding site recognition. In fact, Pettit andco-workers reported no inhibition of tubulin polymerization with thecombretastatin prodrug while only a 40% activity is present for thephenstatin prodrug compared to phenstatin. The IC₅₀ values forinhibition of tubulin polymerization are 1.2±0.1 μM for CA-4,>80 μM forCA-4 prodrug, 1.0±0.2 μM for phenstatin and 21±3 μM for phenstatinprodrug; similar results are expected for the amino-CA-4 8 and thephosphoramidate 10.³¹ The IC₅₀ for the amino-CA-4 8 is 1.2±0.02 μM, andthe phosphoramidate 10 has little if any activity.³² TABLE IIIComparative GI₅₀ Values Against Human Cancer Cell Lines for Amine-CA-48, Amine-CA-4 Prodrug 10. Phenstatin. Phenstatin Prodrug andCombretastatin A-4 Prodrug. GI₅₀, values are reported as concentrationsin μg/mL ND = Not determined,^(a) Data obtained in collaboration withDr. George R. Pettit.^(30b) Data obtained from synthesis of phenstatinphosphate. Amine-CA-4 Phenstatin Combretastatin Cell Type Cell-LineAmine-CA-4 8^(a) Prodrug 10^(a) Phenstatin Prodrug^(b) A-4 Prodrug^(b)Ovarian OVCAR-3 ND 1.9 × 10⁻¹ 2.3 × 10⁻³ 2.5 × 10⁻³ 2.3 × 10⁻² CNSSF-295 ND 2.4 × 10⁻¹ 5.2 × 10⁻² 1.2 × 10⁻² 3.6 × 10⁻² Lung-NSC NCI-H4606.8 × 10⁻⁴ 3.5 × 10⁻¹ 5.7 × 10⁻³ 3.5 × 10⁻² 2.9 × 10⁻² Colon KM20L2 ND2.8 × 10⁻¹ 4.0 × 10⁻⁴ 2.7 × 10⁻¹ 3.4 × 10⁻¹

In terms of in vivo systems, phosphoramidate analog 10 is able toprovide a more soluble compound than the amine 8, thereby incrementingits bioavailability. Under, in vivo biological conditions, the P-N bondcan be broken by serum phosphatases releasing the amine which caninhibit tubulin polymerization in a manner analogous to combretastatin

Anti-Angiogenesis

The growth of a tumor depends on the generation of blood vessels whichwill provide all the metabolites required during cell division. Thedevelopment of anti-angiogenic compounds is especially useful in thetreatment of solid tumors, since these compounds have the potentialcapability of selectively disrupting the vasculature of tumor cellswhile leaving healthy cells in a viable situation. The combretastatinA-4 prodrug has demonstrated anti-angiogenic activity since small dosesof the drug are toxic to tumor vasculature.³⁴ Enhanced cytotoxicactivity was observed against endothelial cells associated with thetumor vasculature of cancerous cells, while at the same time it wasreported to have no effect against other endothelial cells which arelocated distant from the tumor itself.^(34, 35) The mechanism of actionof combretastatin A-4 prodrug, as an anti-angiogenic drug for cancertreatment, is under investigation because the development of bloodvessels is crucial for the survival and growth of solid tumors. Oneproposed mechanism for anti-angiogenesis involves induction of apoptosis(cell suicide) of the cells instead of necrosis. An evaluation of theability of the new phosphoramidate 10, along with structurally similarcompounds, to induce apoptosis of endothelial cells will be undertakenin the near future.

Synthesis of the Phosphoramidate Analog

(Z)- 1 -(3′-Diethylphosphoramidate-4′-methoxyphenyl)-2-(3″, 4″,5″-trimethoxyphenyl)ethene 10.

Diethylchlorophosphite (0.103 g, 0.66 mmol) was dissolved in anhydrousdiethyl ether (2.5 ml) and cooled to −78° C. Diisopropylethyl amine(0.187 g. 1.45 mmol) was dissolved in Et₂O (1.0 ml) and added slowlyover a period of 2 mm to the reaction mixture by syringe. Amino-stilbene8 was dissolved in Et₂O (1.0 mL) and added slowly to the reactionmixture by syringe. The reaction mixture was stirred under nitrogen at−78° C. for 2 hours, followed by stirring for 1 hour at roomtemperature. The mixture was filtered, and the solvent was removed underreduced pressure. A yellow oil was obtained which was dissolved in dryCH₂Cl₂ (5 mL). The oil was cooled to 40° C. and a solution of m-CPBA(0.193 g, 1.12 mmol) in CH₂Cl₂ (5 mL) was added. It was stirred over onehour at room temperature. After this time, the reaction mixture wascooled to −40° C. and filtered through a sintered glass funnel. Theliquid was collected with vigorous stirring over sodium sulfite (5%) (20ml) in order to quench the reaction. The product was isolated byextraction with CH₂Cl₂, and washed with a saturated solution of NaHCO₃.The yellow oil which was obtained was dried over MgSO₄. Purification byflash chromatography (70/30, hexanes/EtOAc) afforded the phosphoramidate10 as a yellow oil (0.130 g, 0.29 mmol, 44%).

'H-NMR (CDCl₃, 360 MHz) δ7.12 (d, J=1.9 Hz, IH, ArH), 6.88 (dd, J=8.4Hz, 2.0 Hz, IH, ArH), 6.72 (dd, J=8.4 Hz, 1.7, IH, ArH), 6.49 (s, 2H,ArH), 6.51 (d, J=12.1 Hz, IH, vinyl CH), 6.41 (d, J=12.1 Hz, IH, vinylCH), 5.67 (d, J=10.0 Hz, NH), 4.02 (m, 4H, CH₂), 3.83 (s, 3H, OCH₃),3.83 (s, 3H, OC₃), 3.68 (s, 6H, OCH₃), 1.25 (t, 6H, J=7.1 Hz, CH₃).

¹³C-NMR (CDCl₃, 90 MHz) δ152.7, 146.7, 146.6, 137.0, 132.7, 130.3,129.7, 129.1, 129.0, 122.0, 117.0, 109.9, 106.0, 62.8, 60.7, 55.7, 16.1.

³¹P-NMR (CDCl₃, 145 MHz) δ0.84.

HRMS (EI) M+calcd for C₂₂H₃₀N0₇P 451.1760, found 451.1765.

EXAMPLE I Synthesis of the Indole-Based Anti-Tubulin Agents

Preparation of 2-Phenyl Indole 31

Method I (2 steps);

To a well-stirred solution of KOH (0.926 g, 16.5 mmol) in EtOH (18 ml)and H₂O (9 ml) at rt was added m-anisidine (2.192 g, 17.80 mmol) bysyringe. The solution was then stirred at 0° C. After 10 min, thesolution of 2-bromo-4-methoxyacetophenone (4.09 g, 17.80 mmol) was addeddropwise with an addition funnel over a 40 minute period. After 24 h, 0°C. to rt, water was added. The product was isolated by extraction (I HHCI, NaHCO3, brine, MgSO4). The product was purified byrecrystallization (50:50 EtOAc:hexanes) to afford secondary amine 30(2.46 g, 9.07 mmol, 52%) as yellow solid.

'H NMR (CDCI₃): δ7.98 (2H, D, J=8.9 Hz), 7.12 (IH, t, J 8.1 Hz), 6.97(2H, d, J 8.9 Hz), 6.30 (3H, m), 4.54 (2H, s), 3.88 (3H, s), 3.79 (3H,s).

Polyphosphoric acid (PPA) was charged to around-bottom flask and thetemperature was raised to 80° C. with vigirous stirring. To this flaskwas added the foregoing amine 30 (4.0 g, 14.74 mmol) in 6 portions overa 30 minute period. After 2 h, 80° C. to 90° C., water was added. Theproduct was isolated by extraction ( EtOAc, NaHCO₃, brine, MgSO₄).Purification by recrystallization (acetone) afforded indole 31(0.544 g,2.15 mmol, 15%) as a pale yellow solid.

'H NMR (CDCl₃): δ11.24 (IH, br, s), 7.72 (2H, d, J 8.82 Hz), 7.36 (IH,d, J=8.57 Hz), 7.00 (2H, d, J=8.84 Hz), 6.85 (IH, d, J=2.07 Hz), 6.66(IH, d, J=1.66 Hz), 6.63 (IH, dd, J 8.59, 2.28 Hz), 3.78 (3H, s), 3.77(3H, s).

¹³C NMR (CDCl₃): δ158.15, 155.22, 137.44, 136.33, 125.60, 124.93,122.82, 120.04, 114.07, 109.00, 96.97, 94.01, 54.93, 54.88.

Method 2(1 step):

To a boiling mixture of in-anisidine (1.56 ml, 20.0 minol) andN,N-dimethylaniline (3.5 ml) was added 2-bromo-4-methoxyacetophenone(1.37 g in EtOAc, 6.00 mmol) slowly by syringe. After addition, themixture was kept at 170° C. for 1 hour. The reaction mixture was cooledto room temperature and a dark colored solid was formed. EtOAc was addedalong with HCI (2 N). The aqueous layer was extracted with EtOAc severaltimes. The combined organic layers were washed with brine, and driedover MgSO₄. Solvent was removed under the reduced pressure to afford adark brown colored solid. Purification by recrystallization in EtOHafforded indole 31 as a white crystalline material.

'H NMR(CDCl₃): δ11.24 (IH,br,s),7.72(2H,d, J 8.82 Hz), 7.36 (IH,d, J8.57 Hz),7.00 (2H,d, J=8.84 Hz), 6.85 (IH, d, J=2.07 Hz), 6.66 (IH, d,J=1.66 Hz), 6.63 (IH, dd, J8.59, 2.28 Hz),3.78 (3H, s), 3.77 (3H, s).

¹³C NMR (CDCl₃): δ158.15, 155.22, 137.44, 136.33, 125.60, 124.93,122.82, 120.04, 114.07, 109.00, 96.97, 94.01, 54.93, 54.88.

Melting Point: 208-229.5° C.

HRMS (El) M+calcd for CH₁₆N0₂ 253.3035, found 253.1060.

Preparation of Trimethoxybenzoate 2-Phenylindole 33

To a well stirred solution of indole 31(0.502 g, 1.98 mmol) ino.dichlorobenzene (10 ml) was added trimethoxybenzoylchloride (0.692 g,3.00 mmol). The reaction mixture was heated to reflux for 12 hours.Solvent was removed by distillation under reduced pressure. Aftercooling down to room temperature, a dark solid formed which wasdissolved in chloroform and purified by silica gel column chromatographywith chloroform as the eluent. The collected mixture was again purifiedby column chromatography (50:50 hexanes:EtOAc) affordingtrimethoxybenzyl indole 33 (0.744 g, 1.66 mmol, 84%) as a yellow oilygel. Pale yellow-green crystals were obtained by recrystallization froma mixture of ethanol and hexanes.

'H NMR (CDCl₃): δ8.63 (IH, br, s), 7.88 (IH, d, J=9.39 Hz), 7.24 (2H, d,J=8.78 Hz), 6.95(2H, s), 6.90 (2H, m), 6.71 (2H, d, J=8.79 Hz), 3.86(3H, s), 3.80 (3H, s), 3.73 (3H, s), 3.68 (6H, s);

¹³C NMR (CDCl₃): δ192.23, 159.73, 157.06, 152.42, 142.85, 141.01,136.41, 134.65, 130.16, 124.28, 122.94, 122.17, 113.67, 112.46, 111.52,107.24, 94.54, 60.78, 55.92, 55.54, 55.14.

Melting Point: 153-155° C.

Anal. Calcd for C₂₆H₂₅N0₆: C, 69.79; H, 5.63; H, 3.13. Found: C, 69.61;H, 5.63; N, 3.01.

EXAMPLE 2 Inhibition of Tubulin Polymerization Assay

IC₅₀ values for tubulin polymerization were determined according to theprocedure described in Bai et al. Purified tubulin is obtained frombovine brain cells as described in Hamel and Lin. Various amounts ofinhibitor were preincubated for 15 minutes at 37° C. with purifiedtubulin. After the incubation period, the reaction was cooled and GTPwas added to induce tubulin polymerization. Polymerization was thenmonitored in a Gilford spectrophotometer at 350 nm. The final reactionmixtures (0.25 ml) contained 1.5 mg/ml tubulin, 0.6 mg/mlmicrotubule-associated proteins (MAPs), 0.5 mM GTP, 0.5 mlM MgCl₂, 4%DMSO and 0.1M 4-morpholineethanesulfonate buffer (MES, pH 6.4). IC₅₀ isthe amount of inhibitor needed to inhibit tubulin polymerization 50%with respect to the amount of inhibition that occurs in the absence ofinhibitor. The IC₅₀ value determined for 3-(3′,4′,5′-trimethoxybenzoyl)-2-(4′-methoxyphenyl)-6-methoxyindole was0.5-1.5 μM.

EXAMPLE 3 Cytotoxic Assay with P388 Leukemia Cells

One of the newly prepared compounds was evaluated for cytotoxic activityagainst P388 leukemia cells using an assay system similar to theNational Cancer Institute procedure described below and in Monks et al.The ED50 value (defined as the effective dosage required to inhibit 50%of cell growth) of 3-(3′,4′,5′trimethoxybenzoyl)-2-(4′-methoxyphenyl)-6-methoxyindole was found to be0.0133 μg/mL.

EXAMPLE 4 Growth Inhibitory Activity Against Other Cancer Cell Lines

3-(3′,4′,5′-Trimethoxybenzoyl)-2-(4′-methoxyphenyl)-6-methoxyindole wasevaluated in terms of growth inhibitory activity against several humancancer cell lines, including pancreas, ovarian, CNS, lung-NSC, colon,and prostate lines. The assay used is described in Monks et al. Briefly,the cell suspensions, diluted according to the particular cell type andthe expected target cell density (5,000-40,000 cells per well based oncell growth characteristics), were added by pipet (100 μl) to 96-wellmicrotiter plates. Inoculates were allowed a preincubation time of 24-28hours at 37° C. for stabilization. Incubation with the inhibitorcompounds lasted for 48 hours in 5% CO₂ atmosphere and 100% humidity.Determination of cell growth was done by in situ fixation of cells,followed by staining with a protein-binding dye, sulforhodamine B (SRB),which binds to the basic amino acids of cellular macromolecules. Thesolubilized stain was measured spectrophotometrically. The results ofthese assays are shown in Table 1. Gl₅₀ is defined as the dosagerequired to inhibit tumor cell growth by 50%. TABLE IV Activity ofIndole Ligand Against Selected Human Cancer Cell lines (In Vitro).Indole-based Ligand 33 CELL TYPE CELL LINE GI₅₀ (μG/mL) Pancreas-aBXPC-3 2.0 × 10⁻³ Ovarian OVCAR-3 2.4 × 10⁻³ CNS SF-295 2.4 × 10⁻³Lung-NSC NCI-H460 2.6 × 10⁻³ Colon KM20L2 1.7 × 10⁻³ Prostate DU-145 2.3× 10⁻³

Indole and indole containing compounds, of therapeutic efficacy havebeen known for many, many years. What is truly unique about the indolecompounds described in this application is the fact that these compoundsare the first (to the best of our knowledge) indole-based ligands toincorporate the 3,4,5-trimethoxyaryl motif reminiscent of colchicine andcombretastatin A-4 arranged in an appropriate molecular conformationsuch that a pseudo aryl-aryl pi stacking interaction can take place. Itis our contention that such an aryl-aryl interaction of the appropriatecentroid-to-centroid distance (approximately 4.7Å) is imperative forenhanced binding affinity to the colchicine site on β-tubulin. It isthis binding that ultimately leads to an inhibition of tubulinpolymerization which manifests itself as a cytotoxic event. It should bereadily apparent to any practitioner skilled in the art that there arevarious ways of appending trimethoxyaryl and trimethoxyaroyl groupsaround an indole molecular scaffold in a manner which will result in asimilar molecular conformation capable of undergoing pseudo pi-pistacking. In addition, although the trimethoxyaryl motif seems optimalfor enhanced tubulin binding, it is also very possible that anothercombination of alkoxy substituents (such as ethoxy, propoxy, isopropoxy,allyloxy, etc.) either as a trisubstituted pattern or as disubstituted(with one type of alkoxy moiety) and monosubstituted (with a differentalkoxy moiety), or with three distinct types of alkoxy moieties may alsohave good tubulin binding characteristics. It is also conceivable thatinstead of having aryl alkoxy groups, it may be possible to substitutesimply aryl-alkyl and aryl-alkenyl moieties and still maintain theenhanced cytotoxicity profile. Phenolic groups may also have activity onthese described indole ligands. The synthesis of any of these modifiedindole-ligands will be very straight-forward for anyone skilled in theart, and often will only involve a different choice of initial startingmaterials. To prepare these alternative ligands, the same syntheticschemes (FIGS. 6, 9, 11, 12-16), or similar schemes with only slightmodifications may be employed. In previous studies with thebenzo[b]thiophene ligands, we have demonstrated that the carbonyl groupcan be replaced with an oxygen to generate a new compound whichmaintains the same or similar biological efficacy with tubulin.Similarly, the replacement of the carbonyl group in the described indoleligand may be replaced with an oxygen atom (ether linkage) to generate anew derivative which would be predicted to have good activity withtubulin. This compound may be prepared by an addition eliminationreaction utilizing the trimethoxyphenolic anion as a nucleophile asdescribed by us for the benzo[b]thiophene compounds. Other linkage atomsbetween the aryl aryl rings are conceivable as well.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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1. A compound of the structure:

wherein R¹ through R⁵ contain at least one phenolic moiety or at least one amine group (NH₂, NHR¹, or NR⁶R⁷ where R⁶ and R⁷ are the same or different alkyl having up to 8 carbon atoms), benzyl, or aryl while the remaining R¹ through R⁵ are hydrogen.
 2. A compound of the structure:

wherein R¹ through R⁵ contain at least one phosphate ester moiety (-OP(O)(O⁻M⁺)₂) or a phosphoramidate (-NP(O)(O⁻M⁺)₂) where M is a cation or (-NP(O)(OR)₂) where R is an alkyl with up to 8 carbon atoms (the two R groups are the same or different, benzyl, or aryl while the remaining R¹ through R⁵ are hydrogen.
 3. A compound of the structure:

wherein R¹ through R⁵ contain at least one phosphate ester moiety (-OP(O)(O⁻M⁺)₂) or a phosphoramidate (-NP(O)(O⁻M⁺)₂) where M is a cation or (-NP(O)(OR)₂) where R is an alkyl with up to 8 carbon atoms (the two R groups are the same or different), benzyl, or aryl while the remaining R¹ through R⁵ are hydrogen, and R⁶ is hydrogen or alkyl.
 4. A compound of the structure:

wherein R¹ through R⁵ contain at least one phenolic moiety or at least one amine (NH₂, NHR¹, or NR⁶R⁷ where R⁶ and R⁷ the same or different alkyl having up to 8 carbon atoms, benzyl, or aryl groups) while the remaining R¹ through R⁵ are a hydrogen.
 5. A compound of the structure:

wherein R¹ through R¹ contain at least one phosphate ester moiety (-OP(O)(O⁻M⁺)₂) or a phosphoramidate (-NP(O)(O⁻M⁺)₂) where M is a cation or (-NP(O)(OR)₂) where R is an alkyl with up to 8 carbon atoms (the two R groups are the same or different), benzyl, or aryl while the remaining R¹ through R⁵ are hydrogen.
 6. A compound of the structure:

wherein R¹ through R⁵ contain at least one phosphate ester moiety (-OP(O)(O⁻M⁺)₂) or a phosphoramidate (-NP(O)(O⁻M⁺)₂) where M=a cation or (-NP(O)(OR)₂ ) where R is an alkyl with up to 8 carbon atoms (the two R groups are the same or different), or benzyl, or aryl groups, while the remaining R¹ through R⁵ are a hydrogen and R⁶ is hydrogen or alkyl.
 7. A compound of the structure:

wherein R¹ through R⁵ contain at least one phenolic moiety or at least one amine group (NH₂, NHR or NR⁶R⁷ where R⁶ and R⁷ are the same or different alkyl having up to 8 carbon atoms may be the same or different), or benzyl, or aryl groups) while the remaining R¹ through R⁵ are a hydrogen.
 8. A compound of the structure:

wherein R¹ through R⁵ contain at least one phosphate ester (-OP(O)(O⁻M⁺)₂) or a phosphoramidate (-NP(O)(O⁻M^(+6l )) ₂) where M is a cation or (-NP(O)(OR)₂) where R is an alkyl with up to 8 carbon atoms (the two R groups are the same or different), benzyl, or aryl while the remaining R¹ through R⁵ are hydrogen.
 9. A compound of the structure:

wherein R¹ through R⁵ contain at least one phosphate ester (-OP(O)(O⁻M⁺)₂) or phosphoramidate (-NP(O)(O⁻M⁺)₂) where M is a cation or (-NP(O)(OR)₂) where R is an alkyl with up to 8 carbon atoms (the two R groups are the same or different), benzyl, or aryl, while the remaining R¹ through R⁵ are hydrogen, and R⁶ is hydrogen or alkyl.
 10. A compound of the structure:

wherein R¹ through R⁵ contain at least one phenolic moiety or at least one amine group (NH₂, NHR¹, or NR⁶R⁷ where R⁶ and R⁷ are the same or different alkyl having upt to 8 carbon atoms, benzyl, or aryl) while the remaining R¹ through R5 are a hydrogen.
 11. A compound of the structure:

wherein R¹ through R⁵ contain at least one phosphate ester (-OP(O)(O⁻M⁺)₂) or a phosphoramidate (-NP(O)(O⁻M⁺)₂) where M is a cation or (-NP(O)(OR)₂) where R is an alkyl with up to 8 carbon atoms (the two R groups are the same or different), benzyl, or aryl, while the remaining R¹ through R⁵ are hydrogen.
 12. A compound of the structure:

wherein R¹ through R¹ contain at least one phosphate ester moiety (-OP(O)(O⁻M⁺)₂) or a phosphoramidate (-NP(O)(O⁻M⁺)₂) where M is a cation or (-NP(O)(OR)₂) where R is an alkyl with up to 8 carbon atoms (the two R groups are the same or different), benzyl, or aryl while the remaining R¹ through R⁵ are hydrogen, and R⁶ is hydrogen or alkyl.
 13. A compound of the structure:


14. A compound of the structure:


15. A compound of the structure:


16. A compound of the structure:


17. A compound of the structure:


18. A compound of the structure:


19. A compound of the structure:


20. A compound of the structure:


21. A compound of the structure:


22. A compound of the structure:


23. A compound of the structure:


24. A compound of the structure:


25. A compound of the structure:


26. A compound of the structure:


27. A compound of the structure:


28. A compound of the structure:


29. A compound of the structure:


30. A compound of the structure:


31. A compound of the structure:


32. A compound of the structure:


33. A compound of the structure:


34. A compound of the structure:


35. A compound of the structure:


36. A compound of the structure:


37. A compound of the structure:


38. A compound of the structure:

wherein R is chosen to be any appropriate alkyl or branched alkyl having up to 8 carbon atoms, the two R groups may be the same or different.
 39. A compound of the structure:


40. A compound of the structure:

wherein M+is a cation.
 41. A method for inhibitiing tubulin polymerization by contacting a tubulin-containing system with an effective amount of a compound described in any of claims 1-40.
 42. The method of claim 41 wherein said system is in a tumor cell.
 43. A method of treating a host afflicted with a neoplastic disease by administering to said host a compound described in any of claims 1-40.
 44. The method of claims 41, wherein the contacted system is located in a patient.
 45. The method of claim 41 described further as for treating cancer, wherein said cancer may be chosen from the group containing leukemia, lung, colon, thyroid, CNS, melanoma, ovarian, renal, prostate, and breast cancers.
 46. A preparation for pharmaceutical use containing a compound from any of claims 1-40 as an active component along with a pharmaceutically acceptable carrier.
 47. A method for selectively targeting and destroying tumor vasculature comprising administering an effective amount of a compound described in any of claims 1-40. 